PKG II reverses HGF-triggered cellular activities by phosphorylating serine 985 of c-Met in gastric cancer cells

Abstract

Previous studies showed that type II cGMP-dependent protein kinase G (PKG II) could inhibit the activation of epidermal growth factor receptor (EGFR). Both c-Met and EGFR belong to family of receptor tyrosine kinases (RTKs) and have high molecular analogy. However, the effect of PKG II on c-Met activation is unclear. This study was designed to investigate the inhibitory effect of PKG II on the activation of c-Met and consequent biological activities. The results from CCK8 assay, Transwell assay and TUNEL assay showed that HGF enhanced cell proliferation and migration, and decreased cell apoptosis. Activated PKG II reversed the above changes caused by HGF. Immunoprecipitation and Western blotting results showed that PKG II could bind with c-Met and phosphorylate its Ser985, and thereby inhibited HGF-induced activation of c-Met and MAPK/ERK and PI3K/Akt/mTOR mediated signal transduction. When Ser985 of c-Met was mutated to Alanine for preventing phosphorylation of this site, the blocking effect of PKG II on c-Met activation was annulled. When Ser985 of c-Met was mutated to Aspartic acid for mimicking phosphorylation of this site, HGF-induced activation of c-Met was prevented. In conclusion, the results indicated that PKG II could block c-Met activation via phosphorylating Ser985 of this RTK.

Keywords: c-Met; gastric cancer cell; inhibition; phosphorylation; type II cGMP-dependent protein kinase G.

Figure 1. Analysis of the effect of PKG II on HGF-induced changes of proliferation, migration and apoptosis activities

(A) The expression of c-Met in human gastric epithelial cell line GES-1 and gastric cancer cell lines AGS, HGC-27, SGC-7901 and BGC-823. (*P < 0.05, compared to GES-1 cell). (B–G) The detection of proliferation, migration, apoptosis activities and related protein expression. The AGS and HGC-27 cells were infected with either Ad-LacZ or Ad-PKG II, serum-starved for 12 h, stimulated with 8-pCPT-cGMP (100 or 250 μΜ) for 1 h, and then treated with HGF (50 ng/ml) for 24–48 h. (B) The relative proliferation activities were presented. (C) The expression of PCNA was detected by Western blotting with an anti-PCNA antibody. (D) The transwell migration assay was applied to detect the migration activity. The representative data showed the relative activities of migration. (E) The expression of MMP7 was detected by Western blotting with an anti-MMP7 antibody. (F) TUNEL method was applied to analyze the apoptosis activity, and the average ratio of apoptotic cells per field (magnification, × 200) was shown. (G) Detection of Bax and Bcl-2 protein by Western blotting in different groups. (*P < 0.05, compared to LacZ group; ^#P < 0.05, compared to PKG II group; ^&P < 0.05, compared to LacZ+HGF group, or PKG II+HGF group).

Figure 2. Analysis of the effect of PKG II on the activation of main components in MAPK/ERK pathway induced by HGF

The AGS and HGC-27 cells were treated same as in Figure 1, excepting that the HGF (50 ng/ml) treatment is 10 min. (A) The phosphorylation of ERK1/2 in different group was detected by Western blotting with antibody against p-ERK1/2 (Thr 202/Tyr 204). (B) The phosphorylation of MEK1/2 in different group was detected by Western blotting with antibody against p-MEK1/2 (Ser217/221). (*P < 0.05, compared to LacZ group; ^#P < 0.05, compared to PKG II group; ^&P < 0.05, compared to LacZ+HGF group, or PKG II+HGF group).

Figure 3. Analysis of the effect of PKG II on the activation of main components in PI3K/Akt/mTOR pathway induced by HGF

The AGS and HGC-27 cells were treated with the same conditions as in Figure 2. (A) Western blotting was performed to detect the phosphorylation of PI3K p85 at Tyr 458 site in different groups. (B) The phosphorylation of Akt were detected by Western blotting with antibody against p- Akt (Thr 308). (C) Western blot analysis of Ser2448 phosphorylation of mTOR in different groups. (*P < 0.05, compared to LacZ group; ^#P < 0.05, compared to PKG II group; ^&P < 0.05, compared to LacZ+HGF group, or PKG II+HGF group).

Figure 4. Analysis of the effect of PKG II on Tyr1234 phosphorylation of c-Met stimulated by HGF

The AGS and HGC-27 cells were treated with the same conditions as in Figure 2. (A) The Tyr1234 phosphorylation level of c-Met in HGC-27 cells were detected by Western blotting and the representative data was shown. (B) Western blot analysis of Tyr1234 phosphorylation of c-Met in different groups of AGS cell.

Figure 5. PKG II blocks HGF-induced c-Met activation by phosphorylating c-Met at Ser985

(A) Interaction between PKG II and c-Met. The AGS and HGC-27 cells were infected with Ad-PKG II, and then successively stimulated with 8-pCPT-cGMP (250 μΜ, 1 h) and HGF (50 ng/ml, 10 min). The cells were lysed and the lysates were immuno-precipitated with anti-c-Met antibody, anti-PKG II antibody and isotype-matched IgG, respectively. The precipitates were probed with anti-PKG II antibody (or anti-c-Met antibody), with 5% of cell lysate as protein input control. (B) PKG II increased pan-ser/thr phosphorylation of c-Met. The cells were infected with Ad-PKG II and then stimulated with 8-pCPT-cGMP (250 μΜ, 1 h). The cells were lysed and the lysates were immuno-precipitated with anti-c-Met antibody, and the precipitates were probed with anti-pan-Ser/Thr antibody. (C) PKG II induced Ser985 phosphorylation of c-Met. The cells were treated same as in panel B, and the precipitates were probed with anti-p-c-Met (Ser985) antibody. (D) The sequencing data of plasmid constructs c-Met-WT, c-Met-S985A and c-Met-S985D. (E) Ser985 was a PKG II-specific phosphorylation site in c-Met. The COS-7 cells were transfected with GFP-tagged plasmids of wild type c-Met or mutant c-Met-S985A, and then successively infected by Ad-PKG II for 24 h and treated with 8-pCPT-cGMP (250 μΜ) for 1 h. The cells were harvested and lysate was prepared. Immunoprecipitation with antibody against GFP was performed to precipitate wild type or mutant c-Met, and the pan-Ser/Thr phosphorylation in the precipitates were analyzed by Western blotting with anti-pan-Ser/Thr antibody. (F) HGF-induced Tyr1234 phosphorylation of mutant c-Met-S985A is not inhibited by PKG II. The COS-7 cells were treated as the above and then stimulated by HGF (50 ng/ml) for 10 min. The cell lysates were immuno-precipitated with anti-GFP antibody, and the precipitates were probed with anti-p-c-Met (Tyr 1234) antibody. (G) The COS-7 cells were treated same as in panel (F) The results showed that HGF-induced Tyr1234 phosphorylation was inhibited in c-Met-WT and c-Met-S985D, while not inhibited in mutant c-Met-S985A.